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(New page: == Media composition (week 2) == '''Paper 1''' http://iai.asm.org/cgi/reprint/56/9/2430.pdf This paper is on the different expression of genes in E.coli in iron-rich and iron-deficie...)
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Revision as of 10:03, 29 July 2008
Media composition (week 2)
This paper is on the different expression of genes in E.coli in iron-rich and iron-deficient media and they were shifting the bacteria from high-iron to low-iron content in the expt. Their media prepared as follows:
Protocol: Cells of E. coli were routinely grown in Luria broth (LB) at 37°C with shaking LB was supplemented with appropriate antibiotics at the following concentrations: ampicillin, 50 ,ug/ml; and chloramphenicol, 30 jig/ml LB containing 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride per liter was used as the iron-rich medium Deferrated LB was prepared by treating LB with 10 g of chelex 100 (Bio-Rad Laboratories, Richmond, Calif.) per liter for 4 h The deferrated LB was decanted, and residual chelex 100 was removed by filtration. The deferrated LB was transferred to acid-washed Pyrex bottles and sterilized by autoclaving A single large batch of deferrated medium was prepared, stored at 4°C, and used throughout the study I think we can probably try to follow their protocol in preparing the deferrated LB medium then increase the iron content accordingly as we wish... Iron can be supplemented in the form of iron citrate...We can prepare a stock solution of iron citrate (1:100 iron to citrate ratio) following the protocols on the paper printed on Friday...
Iron citrate stock solution protocol: A stock solution of iron citrate (iron-to-citrate ratio, 1:100) was made by dissolving ferrous sulfate (final concentration, 4 mM) in sodium citrate (final concentration, 400 mM) pH is adjusted to 7 with NaOH Sodium citrate was prepared in an identical way, but the ferrous sulfate was omitted for the control i.e. iron free medium The only concern I have is whether we need to remove absolutely all iron and if the deferration method is removing all iron ions or just certain ion...?! Another iron-removing protocol is also mentioned in this second paper so we can pick one to use...or maybe ask Duncan first :-/
"The iron concentration of L broth was reduced by extraction with 8-hydroxyquinoline by the method of Pugsley and Reeves J Bacteriol. 1976;127:218–228."
Anaerobic growth protocol...if needed: Anaerobic fermentative growth was performed in 15-ml optically matched glass tubes filled to the top with L broth plus 0.5% glucose, sealed with Subaseal caps, and incubated at 37°C in a water bath without shaking Anaerobic respiratory conditions were identical, except that either 40 mM sodium fumarate or 40 mM sodium nitrate was added to the medium.
This paper is on the regulation of the iron-dependent expression of some genes in E.coli by comparing cultures in iron-supplemented and normal medium...
Here is the protocol they have indicated in the paper which I think is not as appropriate as the one above...??? The LB medium contained 10 g Bactotryptone,5 g yeast extract, and 10 g NaCl per l and was adjusted to pH 7.0 with ~1.5 g of K2HPO4 The M9CA medium consisted of minimal A salts (Maniatis et al., 1982), 0.2% casamino acids, 0.2% glucose, 3 mg pantothenate, and 5 mg of thiamine per litre. The strains were grown overnight at 37oC (with shaking in the air) in a LB medium containing the required antibiotics The overnight cultures were diluted 200-fold into a M9CA medium and grown to a density of A600 = 0.6 − 0.8 The nice thing is that this paper has mentioned the iron assay (flame test thingie) as well as the result for the iron concentration within E.coli in the iron-supplemented medium!
Iron assay: The iron content of the cell-free extracts was measured using a flame atomic absorption spectrophotometer (Varian Spectra AA 400, Minasco Australia Pty Ltd., Sunbury, Australia) with a deuterium background corrector The samples were diluted 1 : 5 with de-ionized water Seronorm 103 serum standard (Nycomed, Oslo, Norway) was used for the standardization of the element analysis and the mean (n = 7) concentration of iron deviated −3% from the certified value As a reference material, bovine liver standard 1577a (National Institute of Standards and Technology, Gaithersburg, USA) was used for validation of the analytical methods. The analytical value (n = 7) for iron deviated −1.3% from the certified value Result: "E. coli, when grown in an iron-enriched medium, contained about four fold more iron (6.1 ± 0.6 ng iron/mg protein) compared to the cells that were grown in a normal M9CA medium (1.5 ± 0.3 ng iron/mg protein)." So according to this paper they've got a 4 fold increase in iron concentration in E.coli...! Don't know if that'll be enough...